Medicament and method for inducing immunity to infectious bovine keratoconjunctivitis

ABSTRACT

A medicament and method for inducing immunity in to infectious bovine keratoconjunctivitis in cattle. The medicament comprises the gram negative cocci Neisseria or Branhamella which are non-etiological agents of infectious Keratoconjunctivitis yet unexpectedly are found to afford an immunity to infectious bovine keratoconjunctivitis when administered to cattle.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to prophylactic treatment and inducingimmunity of infectious bovine keratoconjunctivitis which is a disease ofthe eyes of cattle commonly called Pinkeye caused by the bacteriaMoraxella bovis. More particularly, the present invention relates tomedicament or medicines and methods used in such treatment.

2. Description of the Prior Art

Pinkeye is a highly contagious disease of the eyes of cattle. Thedisease is characterized by an acute to chronic inflammation of the eyeand impairs the sight of the animal. It affects cattle of all ages andbreeds and is sufficiently debilitating to cause enormous financial lossin the cattle industry.

In the past, it has been discovered that pinkeye is caused by thebacteria Moraxella bovis. In an effort to provide prophylactic treatmentfor pinkeye, various viable and non-viable Moraxella bovis treatmentshave been prepared. It was thought that by means of varying the methodof introduction or the method of attenuation of the Moraxella bovis,bacteria antibodies sufficient to provide immunity to pinkeye could becreated in cattle. However, results from these efforts have not beenentirely satisfactory. Apparently, introduction of Moraxella bovis doesnot create an immunity to itself in a manner which is either longlasting or effective for all cattle. Further, while infection in one eyeof an animal may cause an immunity in that particular eye, theunaffected eye is not immunized and the same animal may be infected inthe unaffected eye at a later time.

Treatment of diseased animals is somewhat impractical. It is difficultto administer medical treatment to a diseased animal in a range herdwhich is the most common location of the diseased animal. Moreover,methods of disease treatment which require special apparatus forimmobilizing the animals head are difficult to utilize in the field.Curative results of treatment in an animal hospital are also notsatisfactory. Accordingly, prophylactic treatment and immunization isthe most practically beneficial method of treating pinkeye.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a medicament forinducing immunity to infectious bovine keratoconjunctivitis.

It is also an object of the present invention to provide an improvedmethod of inducing immunity to infectious bovine keratoconjunctivitis.

In accordance with these objects, the present invention comprises amedicament for inducing immunity to infectious bovinekeratoconjunctivitis. This medicament comprises an amount ofgram-negative cocci selected from the genera of the family Neisseriaceaenot including Moraxella effective to induce immunity to infectiouskeratoconjunctivitis. Preferably, the gram-negative cocci are selectedfrom the genera Branhamella and Neisseria and do not include the speciesNeisseria gonorrhoeae and Neisseria meningitidis.

The method of the present invention comprises introducing in cattle aneffective amount of the above described medicament so as to induce animmunity to infectious bovine keratoconjunctivitis. A particularlyappropriate method of introducing the medicament comprises topicalapplication of the gram-negative cocci in an appropriate carrier to aneye to be immunized.

Antibodies induced by the pinkeye disease or naturally occurringquantities of genera of the family Neisseriaceae do not provide immunityto pinkeye infection of previously undiseased eyes. By the medicament ofthe present invention and the method of treatment of the presentinvention, it has been discovered that bacteria other than the Moraxellabovis bacteria which causes pinkeye can produce antibodies sufficient toimmunize against Moraxella bovis and, therefore, against infectiousbovine keratoconjunctivitis. The medicament and treatment of the presentinvention do not cause any disease in the treated cattle.

For a further understanding of the invention and further objects,features and advantages thereof, reference may not be had to thefollowing description taken in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph illustrating reproduction of pinkeye test resultsrelated to the present invention;

FIG. 2 is a graph illustrating bacterial agglutination titer to M. bovis(tears) test results related to the present invention ;

FIG. 3 is a graph illustrating bacterial agglutination titer to M. bovis(serum) test results related to the present invention;

FIG. 4 is a graph illustrating test results of a test for IgG in tearsrelated to the present invention;

FIG. 5 is a graph illustrating test results of a test for IgA in tearsrelated to the present invention;

FIG. 6 is a graph illustrating test results of M. bovis affected cattlerelated to the present invention; and

FIG. 7 is a graph illustrating test results of Neisseria-Branhamellavaccinated cattle related to the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As shown in the examples and tests set forth below, Moraxella bovis doesnot appear to be an effective immune stimulator. By the presentinvention, it has been discovered that gram-negative cocci of generaselected from the family Neisseriaceae not including Moraxella areeffective immune stimulators and antibodies produced in this immunestimulation are effective to produce an immunity against the infectiousbovine keratoconjunctivitis produced by Moraxella bovis.

It is not yet certain whether the most effective immune response iscreated by cocci selected from the genus Branhamella or the genusNeisseria. It is not thought that an appropriate medicament wouldinclude the species Neisseria gonorrhoeae and Neisseria meningitidis inthat these species are extremely dangerous to humans.

Moraxella bovis is considered to be the main causative agent ofinfectious bovine kertoconjunctivitis (IBK), commonly known as pinkeye.IBK has been reproduced with M. bovis organisms alone and in combinationwith other enhancing factors.

Numerous attempts have been made to produce a pinkeye vaccine utilizingviable and nonviable M. bovis organisms in both experimental and naturalenvironmental conditions. In most cases these vaccines consisted of aheat-killed, formalin-killed, or viable autogenous M. bovis bacteriainjected at weekly intervals intramuscularly or into the third eyelid.While in many cases M. bovis antibodies were produced, fewer positivecultures were obtained, and the severity of lesions were frequentlyreduced, vaccinations did not produce practical protection against thedisease. Other factors such as age, vaccination schedule, and the use ofhomologous stains of M. bovis have been studied.

M. bovis may exist in either a smooth or rough colony form, with roughcolonies exhibiting pili extending from the cell walls. These pili aredelicate elongated unbranched filaments which contain no central poreand have a peritricous distribution. Pili appear to provide additionalextracellular antigens, which may be of importance in development ofresistance to the organism. Studies by others using an M. bovis pilusvaccine have indicated a stimulation of immune response to M. boviswhich may provide a more protective immunity than previous M. bovisvaccines. However, such a vaccine is not as desirable as the vaccine ofthe present invention which does not utilize M. bovis in any viable,non-viable or attenuated form.

Local resistance to bacterial infection of corneal and conjunctivalsurfaces is a complex system involving several antibacterial substances,including secretory IgA, lysozyme, beta-lysin, and lactoferrin. M. bovisantibodies have been found in cattle tears and, while increased duringactive infection, resistance is not necessarily associated with highantibody levels.

In tests described below, IBK was reproduced in calves in 66% ofinnoculated eyes using virulent M. bovis organisms alone. The tests didnot reveal demonstrable alterations in tear or serum levels of IgA, IgGor IgM. Tear antibodies were produced in both affected and non-affectedcalves, but did not appear to provide any protection from the disease.Lysozyme, a potent antibacterial agent in human tears, was not found inany calves.

The purpose of the tests set forth was to examine the clinical andimmunological responses of calves administered a non-virulent bacterialvaccine prior to challenge with a virulent M. bovis and to compare theseresults to those obtained from non-vaccinated calves challenged with thesame organism.

MATERIALS AND METHODS FOR TESTS

Subjects: Twelve week old Holstein calves, free from any clinicaldisease or infection, were used. Prior to use, a thorough examination ofthe anterior segment was performed. All calves were kept inside abuilding in a fly-free environment with no exposure to sunlight.

Test Group: Calves were divided into two groups:

1. Control Group: Six calves (12 eyes) were used as positive controlsand received virulent M. bovis in an effort to produce disease.

2. Vaccinated Group: Twelves calves (24 eyes) were vaccinated with alive non-attenuated bacteria on day 0 and revaccinated on day 14. On day21 these calves were challenged with the virulent M. bovis in a manneridentical to the control group.

Collection of Tears: Tear samples were collected with nonheparinizedcapillary tubes placed in the lower cul-de-sac of the eye followingphysical restraint with a halter and ropes. Approximately 0.5 ml oftears was collected from each eye and placed in the micro-centrifugevials and stored at -40° C.

Determination of disease: All animals were examined daily for thepresence of significant eye diseases, synptoms of which includedblepharospasms, increased lacrimation, conjunctivitis, cornealopacification, ulceration and rupture. Affected eyes were sequentiallyphotographed on a weekly basis.

M. bovis and Neisseria-Branhamella Vaccine Antibody

Production: Four healty New Zealand white rabbits, weighing 8-10 pounds,were used for antibody production against the M. bovis andNeisseria-Branhamella vaccine organisms (2 rabbits each). Each rabbitreceived six separate subcutaneous injections of 0.5 ml of livebacterial in trypticase soy broth (TSB) (8×10⁶ organisms/ml). Theinjections were repeated in five days and blood was taken two weeksafter the second injections, and then at two week intervals. Bloodsamples were centrifuged for ten minutes and the serum removed andfrozen at -40° C.

Bacterial Vaccine Production and Inoculation: A Neisseria-Branhamellabacteria was isolated and grown on 5% BBA for 48 hours at 37° C. usingstandard isolation and growth techniques. Bacterial growth was scrapedfrom plates and suspended in TSB to a concentration of 5×10⁶organisms/ml as determined from BBA plate counts and chamber count. Thissuspension of live bacteria was the vaccine, and was applied topicallyto the eye (0.5 ml/eye) on days 0 and 14 in 12 calves (24 eyes).

The Neisseria-Branhamella bacteria was tested in separate tests todetermine that the bacteria was of the genus Neisseria or the genusBranhamella or both. Results of these tests are set forth in Table I andTable II below.

                  TABLE I                                                         ______________________________________                                        Characteristics of Neisseria-Branhamella                                      used for the Pinkeye Study                                                    ______________________________________                                        1.  Gram - diplococcus                                                            Oxidase +                                                                     Catalase +                                                                2.  Fermentation:                                                             Glucose  Maltose    Lactose  Sucrose  Fructose                                0        0          0        0        0                                           NO.sub.3 Reduction                                                                            Deoxyribonuclease                                             0               0                                                         3.  Growth on Nutrient Agar                                                                       Growth on Brain Heart                                                         Infusion Agar                                             28° C.                                                                          37° C.                                                                            28° C.   37° C.                             +        +          +               +                                         Growth on 5% Bovine Blood Agar Plate                                                 37° C.                                                                 +                                                                      ______________________________________                                         0 = No Fermentation                                                           + = Positive or presence of Growth.                                      

                  TABLE II                                                        ______________________________________                                        Neisseria - Branhamella                                                       ______________________________________                                        1.      Gram-negative cocci .6-.9μ in diameter                             2.      In pairs with adjacent sides flattened                                3.      Oxidase positive                                                      4.      Catalase positive                                                     5.      Non-mobile                                                            6.      Grows at 25° C. on trypticase-soy agar without                         blood-aerobic                                                         7.      Nitrate reduction negative                                            8.      Carbohydrate utilization (C + S):                                             No acid from glucose, lactose, maltose, sucrose,                              xylose, fructose, mannitol, and arabinos                              9.      Urease negative                                                       10.     Phenylalanine negative                                                11.     Citrate utilization negative                                          12.     H.sub.2 S negative                                                    13.     Arginine dehydrolase negative                                         14.     Ornithine decarboxylase negative                                      15.     Lysine decarboxylase negative                                         16.     Indole negative                                                       17.     V-P negative                                                          18.     Gelatin liquefied                                                     ______________________________________                                    

Challenge with Virulent M. bovis: Both the positive control group(challenge only) and the vaccinated group were challenged in anidentical fashion with the same M. bovis organism. A virulent hemolyticM. bovis was grown on 5% BBA for 48 hours, removed from the plates andsuspended in TSB with a concentration of 8×10⁶ organisms/ml. Thisinoculum was applied directly to the eye in one/half of all the calves.

Calves not challenged with the TSB inoculum were challenged with a purebacterial paste. In these calves, the virulent M. bovis was grown for 48hours on BBA. The bacterial growth from one plate was then removed andapplied directly to the eye.

The vaccinated group was challenged on day 21, one week following thesecond vaccination.

Determination of Tear and Serum Antibody Titers to M. bovis andNeisseria-Branhamella by Bacterial Agglutination: Lyophilized samples ofM. bovis and Neisseria Branhamella were reconstituted with 0.3 ml TSBand streaked on three 5% BBA plates. Bacterial organisms (0.5 ml) wereremoved after 48 hours and suspended in 5 ml phosphate buffer solution(PBS) at a pH of 7.2. This suspension was washed three times with PBS.One ml of this bacterial stock solution was diluted with 3 ml of PBS fora 1:4 dilution.

Ten microliters of PBS was placed in each well of a Falcon plate. Tenmicroliters of the sample to be tested was then placed in the first welland a serial twofold dilution was performed. Following this, 10microliters of either the diluted M. bovis and Neisseria-Branhamellabacterial stock solution was placed in all wells. After 24 hours theplates were read for positive agglutination of bacteria.

Determination of Anti-M. bovis Immunoglobulins in Tears: The positivebacterial agglutination reactions for four affected controls and fourvaccinated calves were collected and washed three times with PBS and thebacteria removed. The bacteria were divided into three separate tubes,to which fluorescein conjugated anti-bovine IgA, IgG and IgM were added.Following one hour incubation at room temperature, the bacteria werewashed three times in PBS, placed on a slide and examined forfluorescence with an Olympus U-V microscope. The reaction was graded ona scale of 0 to +4, depending on the degree of fluorescence by twoindependent examiners. In cases of discrepancy, the lower of the twovalues was used.

Determination of General Immunoglobulin Levels in Tears: A single radialimmunodiffusion method was employed for the quantitative determinationof tear immunoglobulins IgA, IgM and IgG. Tear samples and controlstandards were placed in wells and incubated at room temperature for 24hours. After incubation, zones of precipitation were measured and theconcentration determined from a standard curve.

Terminology:

The word infected is defined as "to contaminate with a disease-producingsubstance or agent". All calves were infected with Moraxella bovis. Thecontrol calves were infected (challenged) at day 0, while the vaccinatedgroup was infected (challenged) at day 21 following two vaccinationswith Neisseria-Branhamella at day 0 and day 14.

In the FIGS. the single genus "Neisseria" is used with respect to theNeisseria-Branhamella vaccine produced and tested in the Table I andTable II tests set forth above.

RESULTS OF TESTS Reproduction of IBK

Positive Controls: Six calves (12 eyes) were directly inoculated with0.5 ml virulent M. bovis organisms. While all six calves developedpinkeye, 58% (7 eyes) of the total number of eyes developed significantdisease (FIG. 1). The onset of disease was rapid, with most calvesdeveloping significant pinkeye by day 12. The lesions appeared identicalto the natural disease with progressive corneal ulceration, etc. Boththe bacterial paste and TSB suspension were capable of producingdisease, with the paste producing a more acute and severe process thanthe TSB suspension.

Neisseria-Branhamella Vaccine Group: Twelve calves (24 eyes) werechallenged with virulent M. bovis organisms in a manner identical to thepositive controls, following Neisseria-Branhamella vaccination. None ofthe calves developed any sign of IBK for the 42 days of post challengeobservation.

Bacterial Agglutination Titers to M. bovis

Tears: In the positive control group, both affected and non-affectedeyes developed a similar antibody response which rose to a high ofapproximately 1:75 at three weeks post challenge, then dropped offprecipitously (FIG. 2).

In the vaccinated group, the titers to M. bovis rose above 1:150 beforethe challenge (day 21) and peaked at over 1:200 by day 33 (FIG. 2). Asin the positive control group, the titer then dropped off quickly.

Serum: Titers in both the challenge and control groups remained low forthe first three weeks post challenge (FIG. 3). At that time anincreasing titer was observed in both groups. In the control calves thetiter leveled off after reaching a high of slightly over 1:900. Thevaccinated calves developed a tremendous rise in titer to M. bovis,which was nearly 1:600 in the final sample studied.

Tear Immunoglobins

Three classes of immunoglobulins were studied (IgA, IgG and IgM). TearIgM levels were consistently too low to measure and are not included.

Low levels of IgG were present in baseline tears of both groups (FIG.4). In control calves a slow three-to-five fold increase in tear IgG wasobserved post challenge with little variation between affected andnon-affected eyes. In the vaccinated group, however, tear values werenear 1:225 at the time of challenge. This titer peaked seven daysfollowing challenge at over 1:325 and then dropped off rapidly. Thisrepresented a thirteenfold increase in IgG.

Baseline levels of tear IgA were higher in the control calves than thevaccinated group (FIG. 5). In the control group, both affected andnon-affected eyes demonstrated a slight rise in tear IgA two weeksfollowing challenge, which then fell to below base-line levels.

In vaccinated calves, tear IgA levels increased threefold during thevaccination phase, demonstrated a leveling off phase for three weekspost challenge, then dropped off rapidly.

Classification of Anti-M. bovis Antibodies

Positive Controls: In tear samples collected from four affected controlcalves, antibodies from both the IgA and IgG classes were present,peaking out between two and three weeks post challenge, after which theywere essentially non-detectable (FIG. 6). This was comparable to themild tear titer response seen in the same four calves at days 15 and 21(FIG. 6).

Neisseria-Branhamella Vaccine Group: In the four vaccinated calvesstudied, a greater and more sustained response was seen. By day 21, justprior to challenge, tear titers slightly over 1:600 were associated withhigh levels of IgA and IgG bound to the bacteria (FIG. 7). Following thechallenge there was a marked drop in tear titers, although thefluorescent antibody test demonstrated continued high levels for overthree weeks post challenge.

DISCUSSION

The above tests show it is readily possible to reproduce clinical IBKwith the use of virulent M. bovis organisms alone. The disease wasindistinguishable from the naturally occurring disease in clinicaldevelopment and progression. The Neisseria-Branhamella vaccine providedcomplete protection under the controlled environment of this study.

M. bovis does not appear to be an effective immune stimulator. Theslight rise in tear antibodies was transient and minimal. The lack ofantibody variation in affected and non-affected calf tears suggests theabsence of any meaningful protection against the disease. The minorimmune response was associated with IgA and IgG antibody production,however, these were virtually nondetectable after three weeks postchallenge, and were associated with slight changes in gross IgA and IgGtear levels.

The Neisseria-Branhamella vaccine was in comparison a far superiorstimulator of the immune system against M. bovis antigens. Prior tochallenge, the vaccine stimulated a marked increase in tear M. bovisantibodies, which was concomitant to increasing levels of tear IgA andIgG. These levels were substantially greater than the control group ineither affected or non-affected calves throughout the test period.

Fluorescein antibody studies confirmed that the increased levels of IgAand IgG in tears were, in fact, associated with M. bovis antibodyproduction. While no antibody was detected on day 0, significant levelswere already detectable three days following initial vaccination. At thetime of challenge with M. bovis, these values were much higher than atany time in affected control calves, and remained high for over threeweeks post challenge. In control affected calves, prominent fluoresceinantibody tagging of bacteria was found on days 15 and 21 post challenge,after which they were essentially absent.

Other studies have indicated several cross antigens between theNeisseria-Branhamella vaccine and M. bovis. This apparent crossantigenicity appears to be the basis for the induced protective immunityto M. bovis. This is especially significant since M. bovis does notproduce similar immunity to itself. This lack of auto-immunity may bethe key to both the pathogenicity of M. bovis in bovine cornea and thedifficulty of previous investigators in using M. bovis in various formsas an effective vaccine against itself. On the basis of the above testresults, it is indicated that gram-negative cocci of genera in thefamily Neisseriaceae other than Moraxella are an effective medicament toinduce immunity in cattle to pinkeye. Bacterial agglutination tests ofvarious Neisseria-Branhamella bacteria similar to the bacteria in TablesI and II show variable degrees of ability to produce antibody against M.bovis. The results in Tables I and II were for the bacteria mosteffective in producing antibodies against M. bovis.

It is thought that viable, non-viable and attenuated forms of the generaof the family Neisseriaceae other than Moraxella would be effective forproducing immunity. However, viable forms would be most effective and donot produce any disease in the cattle.

The preferred medicament comprises gram-negative cocci selected from thegenera Neisseria and Branhamella. It is also preferred not to use thespecies Neisseria gonorrhoeae and Neisseria meningitidis since thesespecies are extremely dangerous to humans. It may be preferred to use asingle selected species or only species selected from a single one ofthe genera Neisseria and Branhamella.

Various methods of introducing the medicament to cattle are thought tobe effective to produce immunity to pinkeye. For example, intramuscularinjections or ocular injections are thought to be effective. Oralintroduction and other methods of introduction may also be effective.Topical application to the eye as set forth in the tests is preferred.

Thus, the medicament and method of treatment are well suited to achievethe objects and advantages described as well as those inherent therein.While presently preferred embodiments of the invention have beendescribed for the purpose of this disclosure, various changes can bemade by those skilled in the art which changes are encompassed withinthe spirit of this invention is defined by the appended claims.

The foregoing disclosure and the showings in the examples and drawingsare merely illustrative of the principles of this invention and are notto be interpreted in a limiting sense.

What is claimed is:
 1. A medicament which induces immunity to infectiousbovine Keratoconjunctivitis when administered to cattle comprising: anamount of gram-negative cocci selected form the group consisting of thegenera Neisseria and Branhamella in combination with a pharmaceuticallyacceptable carrier wherein the amount introduced is effective to induceimmunity to infectious bovine Keratoconjunctivitis when administered tosaid cattle.
 2. The medicament of claim 1 wherein said gram-negativecocci are selected from the genus Neisseria.
 3. The medicament of claim2 wherein said cocci from the genus Neisseria are selected from specieswhich are non-pathogenic in cattle.
 4. The medicament of claim 1 whereinsaid gram-negative cocci are selected from species of the generaBranhamella and Neisseria which are non-pathogenic in cattle.
 5. Themedicament of claim 1 wherein said gram-negative cocci are selected fromthe genus Branhamella.
 6. The medicament of claim 1 wherein saidpharmaceutical carrier is suitable for topical application to the eye.7. A method of inducing immunity to infectious bovinekeratoconjunctivitis in cattle comprising:introducing in cattle anamount of gram-negative cocci selected from the Neisseria andBranhamella in combination with a pharmaceutically acceptable carrier;wherein said amount is sufficient to induce immunity to infectiousbovine keratoconjunctivitis in said cattle.
 8. The method of claim 7wherein said gram-negative cocci are of the genus Branhamella.
 9. Themethod of claim 7 wherein said gram-negative cocci are of the genusNeisseria.
 10. The method of claim 9 wherein said gram-negative cocciare selected from species of the genus Neisseria which arenon-pathogenic in cattle.
 11. The method of claim 7 wherein saidintroducing is by topical application to the eyes of cattle to beimmunized.